The chemoreceptors and transport system components of bacteria are among the best-studied membrane proteins and serve as models for the function of hormone and neurotransmitter receptors. Chemotaxis is a virulence factor for pathogenic bacteria, and peptide transport offers an avenue of attack for antibacterial compounds. Bacteria possess two major advantages for analysis of transmembrane receptors at the molecular level. 1) They are readily grown and genetically or biochemically manipulated. 2) Their simple physiology permits direct observation of the phenotypic consequences of receptor mutations. Also, such mutations are rarely lethal. This proposal outlines research that takes advantage of the enormous reservoir of genetic, biochemical, physiological and behavioral data for the enteric bacterium Escherichia coli to examine two chemosensory systems and the transport system associated with one of them. Both chemoreceptors are of the "complex" type. Attractants are first recognized by a substrate-specific binding protein that exists in soluble form in the periplasm between the outer and inner bacterial membranes. the ligand-bound protein interacts with its cognate signal transducer, which spans the inner (cytoplasmic) membrane. this contact evokes an attractant signal from the transducer that modifies the rotation of the flagella to alter the swimming behavior of the bacterial cell. The membrane link of the signalling chain is very intriguing but poorly understood. How are conformation changes induced in the periplasmic domain of a transducer propagated through the membrane to the cytoplasmic domain of the transducer, where the signal to the flagella originates? Maltose and longer chain maltodextrins are recognized by maltose-binding protein (MBP), which interacts with the Tar transducer. Dipeptides consisting of any L-amino acids are recognized by the dipeptide-binding protein (DBP), which interacts with the Tap transducer. Tar in unique in that it binds the attractant L-aspartate as well as MBP; other transducers interact with small attractants or binding proteins, but not both. To fathom how two such disparate molecules as a negatively charged amino acid and a protein of 40,000 daltons can both initiate an attractant signal from one transducer will add substantially to our grasp of the fundamental principles of membrane receptor function. Specifically, mutational analysis and biochemical experiments, especially protein-protein cross-linking, will be used to extend and refine analysis of the MBP/Tar and DBP/Tap interactions. These efforts will be combined with collaborative structural analyses of the proteins. Since it is known how Tar binds aspartate, it is possible to isolate mutants that cannot initiate an attractant signal after aspartate binds. Such mutations will be identified, in part, by their effects in altering the methylation state of the transducers even in the absence of attractants. this information will illuminate the mechanism of transmembrane signalling. The dipeptide permease (Dpp), the uptake system to which DBP belongs, has a potential role in ferrying peptide antibiotics into the bacterial cell. The specificity of the substrate-binding site of DBP will be investigated by mutational analysis and photoaffinity labelling with modified dipeptides. control of dpp operon expression, including its connection to cellular metabolism and the possible roles of regulatory proteins, will also be examined.